Sensing apparatus and method

ABSTRACT

A method of observing reaction intermediaries during a chemical reaction and comprising detecting an electrical signal output from an ion sensitive field effect transistor exposed to said reaction, and monitoring the detected electrical signal to discriminate discrete fluctuations in the electrical signal, the discrete fluctuations indicating reaction intermediaries occurring during a chemical reaction.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application is a continuation of application Ser. No.11/625,844, filed Jan. 23, 2007, now U.S. Pat. No. 8,114,591; which is acontinuation-in-part of patent application Ser. No. 10/471,197, filedMar. 2, 2004, now U.S. Pat. No. 7,686,929; which is the national phaseof International Patent Application No. PCT/GB2002/00965, filed Mar. 11,2002, which claims priority from Patent Application No. GB 0105831.2,filed Mar. 9, 2001; the entire contents of which are hereby incorporatedby reference in this application.

FIELD OF THE INVENTION

The present invention relates to a sensing apparatus and method, andparticularly though not exclusively to a sensing apparatus and methodsuitable for DNA sequence determination. More particularly, it relatesto use of ion-sensitive field effect transistors (ISFETs) to monitorlocal fluctuations in ionic charge corresponding with discrete chemicalevents, especially for example proton release associated with individualnucleotide insertion at the end of an oligonucleotide chain. Monitoringof individual nucleotide insertions by means of a pH sensitive ISFET maybe utilised in DNA sequence determination based on conventional Sangermethod DNA sequence determination and in identifying allelic variants,e.g. single nucleotide polymorphisms (SNPs), relying on detectingextension of oligonucleotide primers designed to target specific nucleicacid sites.

BACKGROUND OF THE INVENTION

DNA sequencing methods have remained largely unchanged in the last 20years [Sterky and Lundeberg, ‘Sequence analysis of genes and genomes’,J. Biotechnology (2000) 76, 1-31]. The Sanger method is a well-knownmethod of DNA sequencing, and comprises DNA synthesis, with terminationof DNA replication at points of di-deoxynucleotide insertion. The DNAsynthesis is followed by electrophoresis of the synthesised DNA toseparate DNA molecules according to their mass to charge ratios, therebyallowing determination of the DNA sequence. A disadvantage of the Sangermethod is that electrophoresis is complex, costly and hazardous. It isan object of the present invention to provide a sensing apparatus andmethod whereby Sanger-type sequence determination employingdi-deoxynucletide triphosphates can be carried out without need forseparation of extended oligonucleotide strands. However, as indicatedabove, the invention can he applied more broadly to monitoring of anychemical event which will give rise to a fluctuation in ionic charge,e.g. proton release.

SUMMARY OF THE INVENTION

According to a first aspect of the invention, there is provided asensing method comprising detecting an electrical signal output from anion sensitive field effect transistor (ISFET), and monitoring thedetected electrical signal to discriminate localised fluctuations ofionic charge, the localised fluctuations of ionic charge occurring at oradjacent the surface of the field effect transistor indicating eventsoccurring during a chemical reaction. More particularly, there isprovided a method of observing reaction intermediaries during a chemicalreaction and comprising detecting an electrical signal output from anISFET exposed to said reaction, and monitoring the detected electricalsignal to discriminate discrete fluctuations in the electrical signal,the discrete fluctuations indicating reaction intermediaries occurringduring a chemical reaction. In a preferred embodiment, said reactionintermediaries arise from one or more nucleotide insertions at the endof a nucleotide chain in a DNA extension reaction (or chain elongation)and individual nucleotide insertions are monitored through detectingchange in the measured electrical signal consequent upon proton releasewith each nucleotide insertion.

The inventors have realised that localised fluctuations of ionic chargewhich occur at the surface of a field effect transistor ay be measured.Although ion sensitive field effect transistors are already known, theyhave previously been used to monitor slow changes of for exampleabsolute values of pH in a reaction mixture as a whole. They have notbeen used to monitor localised fluctuations of ionic charge associatedwith individual chemical events such as nucleotide addition to a DNA. Inknown application of an ion sensitive field effect transistorarrangement, a measurement of the absolute value of the pH of thereaction mixture is made every 30 seconds. Typically, many millions ofchemical reactions will occur between measurements, and this is seen asa change of the absolute value of the pH. The invention allowsindividual chemical events to be monitored.

Preferably, the chemical reaction is DNA synthesis, and the fluctuationsof ionic charge indicate the insertion of individual di-deoxynucleotidetriphosphates (ddNTPs) and deoxynucleotide triphosphates (dNTPs).

A limitation of existing ion sensitive field effect transistorarrangements is that they attempt to measure absolute values of pH andconsequently suffer from drift and hysteresis. The invention monitorsfluctuations of ionic charge rather than absolute values, and thusavoids this problem.

Preferably, the time at which the fluctuations occur and the magnitudeof the fluctuations is monitored to allow sequence determination of DNA.

According to a second aspect of the invention there is provided asensing apparatus comprising an ion sensitive field effect transistorarranged to generate an electrical output signal in response tolocalised fluctuations of ionic charge at or adjacent the surface of thetransistor, means for detecting an electrical output signal from the ionsensitive field effect transistor, and means for monitoring the detectedelectrical signal to discriminate localised fluctuations of ioniccharge, the localised fluctuations of ionic charge indicating eventsoccurring during a chemical reaction.

Again, preferably, the chemical reaction is DNA extension, and thelocalised fluctuations of ionic charge indicate the insertion ofindividual di-deoxynucleotide triphosphates (ddNTP) and deoxynucleotidetriphosphates (dNTP). Preferably, the monitoring means is arranged tomonitor the time at which the localised fluctuations occur and themagnitude of the localised fluctuations, to allow sequence determinationof DNA.

BRIEF DESCRIPTION OF THE DRAWINGS

Specific embodiments of the invention will now be described by way ofexample only with reference to the accompanying figures, in which:

FIG. 1 shows pH changes occurring during pyrophosphate hydrolysis usinga buffered reaction medium;

FIG. 2 is a schematic diagram of a field effect transistor whichembodies the invention;

FIG. 3 is a schematic diagram of a pair of field effect transistorswhich embody the invention;

FIG. 4 is a schematic representation of results obtained using the pairof field effect transistors for DNA sequence determination of the Sangertype on a DNA template employing all required dNTPS and a single ddNTPin the reaction mixture;

FIG. 5 illustrates ISFET sensing apparatus suitable for example forsingle base extension monitoring, e.g. such monitoring for SNPdetection;

FIG. 6 shows DNA templates and primers employed to illustrate use of asensing apparatus of the invention for homozygous SNP detection;

FIG. 7 shows DNA templates and primers employed to illustrate use ofsensing apparatus of the invention for heterozygous SNP detection;

FIG. 8 shows the signals observed with modelling of homzyogous SNPdetection using the templates and primers set out in FIG. 6;

FIG. 9 shows the signals observed with modelling heterozygous SNPdetection using the templates and primers set out in FIG. 7;

FIG. 10 shows signals observed in detecting a nucleotide deletion as anallelic variant in a DNA sequence;

FIG. 11 is a schematic diagram of a microfluidic device containing anembedded ISFET in a low volume reaction chamber as may be employed indetecting DNA extension, including single base primer extension;

FIG. 12 illustrates single base primer extension on a bead as may bemonitored using a device as shown in FIG. 11;

FIG. 13 shows the proton release through DNA extension monitored inapplying the invention to DNA sequence determination;

FIG. 14: ISFET reading with provision of complementary dNTP (dATP) fortemplate as described in Example 1; and

FIG. 15: ISFET reading with provision of non-complementary dNTP (dCTP)for template as described in Example 1.

DETAILED DESCRIPTION

DNA sequencing of the Sanger-type using an embodiment of the inventionis performed as follows: A quantity of DNA of interest is amplifiedusing either a polymerase chain reaction or cloning, and the region ofinterest is primed so that DNA polymerase catalyses DNA synthesisthrough the incorporation of nucleotide bases in a growing DNA chainthereby releasing hydrogen ions, see FIG. 13. This is accompanied invivo with the hydrolysis of pyrophosphate, which at physiological pHleads to the consumption of hydrogen ions [Mathews, Holde, Ahern,Biochemistry, 2nd Edn]; see FIG. 13.

The results shown in FIG. 1 demonstrate the DNA extension reaction andits effect on pH. The pH was measured using a glass electrodearrangement, with measurements of the absolute value of pH taken every30 seconds. The pH can be seen to fall gradually. The embodiment of theinvention uses this reaction to monitor nucleotide insertion, bydetecting localised fluctuations of pH which occur at or adjacent thesurface of an ion sensitive field effect transistor

The ISFET is provided with an ion sensitive silicon nitride layer, ontop of which a layer of polymerase is provided. The release of protonsfrom nucleotide insertion during the DNA extension reaction is detectedby the ISFET, which may or may not be followed by hydrolysis ofpyrophosphate. The magnitude of pH change in either direction (i.e.positive or negative) is detected in order to reliably detect nucleotideinsertion, as described below. Individual nucleotide insertion willoccur approximately every 3 ms at a temperature of 65° C. [Tabor andRichardson, ‘DNA Sequence Analysis with a Modified Bacteriophage T7 DNApolymerase. Effect of pyrophosphorolysis and metal ions’, J. Biol. Chem.(1990) 14, 8322-8328.] The ISFET is able to detect rapid pH changes andhas an immediate response rate measured to be within 1 ms of a pH change[Woias et al., ‘Modelling the short-time response of ISFET sensors’,Sensors and Actuators B, 24-25 (1995) 211-217.]

The hydrolysis of pyrophosphate causes either a net consumption or nochange of hydrogen ions depending on the pH in which the reactionoccurs. In the embodiment of the invention, the reaction is conducted atpH 7. At pH 7 hydrogen ions are overall liberated during nucleotideinsertion. The embodiment of the invention thus monitors drops in pH asindicators of nucleotide insertion.

A pH sensitive FET which embodies the invention is shown in FIG. 2. TheFET is similar to a traditional MOSFET (Metal Oxide Semiconductor FieldEffect Transistor). The FET comprises a silicon oxide dielectric layer1, a silicon nitride chemical sensitive layer 2, and anenzyme/electrolyte interface 3. The layers 1, 2 and interface 3 arelocated between a source 4 and drain 5 (the conventional configurationof a FET). The FET is provided on a silicon chip, which is encapsulatedin epoxy resin to protect it from the reagent mixture. The epoxy resinhelps to protect the FET from hydration and charge migration [Matsuo andEsashi, ‘Methods of ISFET fabrication’, Sensors and Actuators, 1(1981)77-96.] The FET gate itself is not covered by epoxy resin, so that itmay be immersed in the reagent mixture.

The enzyme/electrolyte interface 3 shown in FIG. 2 allows ionsensitivity of the silicon nitride layer 2 to be used for DNAsequencing. The FET functions by producing an exchange of charged ionsbetween the surface of the chemical sensitive layer 2 and the reactingmedium (i.e. the enzyme/electrolyte interface 3):SiOH

SiO⁻+H⁺SiOH₂ ⁺

SiOH+H⁺SiNH3⁺←→SiNH₂+H⁺

The inclusion of silicon nitride is advantageous because it providesincreased and faster sensitivity to changes of pH than would be obtainedin the absence of the silicon nitride. In addition the silicon nitridehelps to protect the FET from hydration and charge migration.

A non-Nernstian response accounts for the immediate sensitivity of theFET, arising from rapid proton dependant binding and unbinding ofcharged ions at the insulating gate silicon nitride surface, whichresults in a reproducible variation in the voltage drop across thesilicon nitride layer 2. The variation of the voltage drop across thesilicon nitride layer 2 correlates with changes of pH. The voltage dropis monitored using instrumentation circuitry, thereby allowing thedetection of individual nucleotide insertions. The measured voltage isreferred to as the flatband voltage.

The enzyme/electrolyte interface 3 is deposited on the silicon nitridelayer using a known enzyme linkage method [Starodub et al.,‘Optimisation methods of enzyme integration with transducers foranalysis of irreversible inhibitors’, Sensors and Actuators B 58 (1999)420-426.] The method comprises silanising the silicon nitride layer 2using aminosilane solution, and then activating the surface usingglutaraldehyde. A drop of buffer/polymerase enzyme solution is thendeposited on the silicon nitride layer 2 and allowed to dry for abouthalf an hour to form the enzyme layer 3.

The embodiment shown in FIG. 2 uses a reference electrode 6 to provide ameasurement of pH changes. The reference electrode is relatively largeand difficult to fabricate. An alternative embodiment of the inventiondoes not use a reference electrode, but instead uses a second FET whichhas the same construction as the first FET, but is provided with anon-enzyme linked layer instead of the enzyme layer 3. Thisconfiguration is advantageous because it provides a differentialmeasurement which gives an improved signal to noise ratio.

The alternative embodiment of the invention is illustrated in FIG. 3.The configuration of this embodiment is based upon a known construction[Wong and White, ‘A Self-Contained CMOS Integrated pH Sensor’, ElectronDevices Meeting IEEE 1988] which has previously been used to monitorgradual slow drift of pH. The embodiment comprises a first operationalamplifier 10 to which the source of the first FET 11 is connected (thefirst FET has the enzyme linked layer), and a second operationalamplifier 12 to which the source of the second FET 13 is connected (thesecond FET has no enzyme linked to the FET). The drains of the first andsecond FETs are connected to a fixed current source (not shown). Outputsfrom the first and second operational amplifiers are passed to adifferential amplifier 14, which amplifies the difference between theoutputs to generate an output signal Vout. Negative feedback from thedifferential amplifier 14 passes to a noble metal electrode 15 which islocated in the reagent mixture. The operational amplifier 14 generatesan output voltage which keeps the voltage applied to the FETs 11,13 thesame despite changes of hydrogen concentration.

The embodiment shown in FIG. 3 is advantageous because it allowsrationalisation of fabrication of the FETs 11,13 and the operationalamplifiers 10,12, 15.

The FETs 11,13 may be arranged to form the first stage of theoperational amplifiers 10,12. This is done for each operationalamplifier by replacing a conventional FET of a long tail pair located atthe input of the operational amplifier, with the first or second FET 11,13. This is advantageous because it allows the first and second FETs toform part of the amplification circuitry.

A schematic example of a Hatband voltage detected using the embodimentshown in FIG. 3 is illustrated in FIG. 4. The example is for an NMOS FETwith the reaction operating in the ion consumption mode, as describedabove (the figure would be inverted for a PMOS FET or if the reactionwas operating in the ion liberation mode). The flatband voltage consistsof pulses representing pH changes associated with nucleotide insertionand drops corresponding to ddNTP insertion and chain termination. Thenumber of local pulses prior to a larger drop determines the number ofbases present before termination at a known base; the magnitude of thelarger drop is dependant on the ratio of ddNTP:dNTP used in the reagentmixture and is important due to the dependence of read length for thatdrop. Through repetition of the process four times in different reactionchambers containing each of the four ddNTPS separately, the completesequence is delineated.

Referring to FIG. 4 in detail, DNA synthesis is performed withtermination of DNA synthesis at points of di-deoxynucleotide insertionof thymine bases. Each individual nucleotide insertion causes theliberation of a hydrogen ion, and these are detected as pulses of theflatband voltage, as can be seen in FIG. 4. When the DNA chain reaches athymine base, nucleotide insertion is prevented for some of the DNAchains, and the amount of hydrogen ion consumption drops leading to adrop in signal output. DNA synthesis continues for those DNA chainswhich were not terminated at the thymine base, and this is seen aspulses of the flatband voltage at the new lower level. The flatbandvoltage falls again when the DNA chain reaches a second thymine base(reflecting the fall in available target due to ddNTP addition), andthen continues to pulse at the lower level.

The method may be used with or without thermocycling. For example,thermocycling may be used to facilitate optimisation, using tagpolymerase as a sequencing enzyme [Alphey, L., ‘DNA sequencing: fromexperimental methods to bioinformatics’ BIOS Scientific Publishers,1997.] The pH of the reagent mixture may be adjusted for example. Adecrease of the pH will lead to the production of more hydrogen ions,but will also tend to inhibit the reaction. Trials have shown pH 6.8 tobe a useful value of pH. Magnesium may be added to the reagent mixtureto actuate the enzyme. The concentrations of the reagents may bemodified. A typical thermocycling sequence is set out in Table 1.

TABLE 1 Cycle Sequencing Temperature Duration Function 95° C. 30 secDenaturing of DNA template 55° C. 30 sec Annealing of primer 72° C. 60sec DNA extension and termination

Operating within a thermal cycler enables multiple repetition of thesequencing process with minimal manipulation. This allows signal tonoise boosting and easier delineation of difficult to read regions suchas GC rich regions or areas of single nucleotide repeats.

Recombinant T7 polymerase may be used instead of taq polymerase. WhereT7 polymerase is used, this may provide increased speed and improvedaccuracy of monitoring nucleotide insertion.

The steps used to fabricate the ion sensitive FET are set out below:

-   PURIFIED SILICON SUBSTRATE-   ADDITION OF DOPANT: PRODUCTION OF p-TYPE SUBSTRATE-   SURFACE OXIDATION: SiO₂ LAYER GENERATION-   SOURCE/DRAIN DEFINITION AND IMPLANTATION-   SILICON NITRIDE DEPOSITION USING LPCVD*-   CONTACT FORMATION-   PASSIVATION-   *Low Pressure Chemical Vapour Deposition

The FETs and in particular those shown in FIG. 3, and the amplificationstages may be replaced or combined with PMOS transistors.

The length of DNA that can be sequenced will normally be limited by thesignal to noise at distal bases as the signal decays with ddNTPinsertion. Using PMOS FETs should allow extension of the read length,but may involve a possible compromise over the location of more proximalbases. Installation of two separate FET circuits, of the type shown inFIG. 3, one NMOS pair of FETs and one PMOS pair of FETs should providethe optimum read length. Biasing in weak inversion is possible, sincethe measurement to be made is of changes to output, rather than absolutevalues, and absolute linearity in signal amplification for signalanalysis is not required.

Measurements may be repeated to provide improved signal to noise ratios.

Since pH sensitive ISFET sensing apparatus can be employed in accordancewith the invention to detect individual nucleotide insertion at the 3′end of an oligonucleotide chain, DNA sequencing in accordance with theinvention extends to embodiments in which single nucleotide extension ismonitored, e.g. such extension of a primer on a template to identifysingle nucleotide polymorphisms (SNPs) in amplified genomic sequences.SNPs are receiving considerable interest due to the fact that many havenow been linked to propensity for various diseases and drug efficacy.SNP detection is thus of interest for disease diagnosis, screening andpersonalised drug therapy. A SNP is defined as variation at a singlebase position generally affecting at least 1% of a defined population.Such a variant may be a substitution, insertion or deletion. AlthoughSNPs do not necessarily cause disease, their association with diseaseand with effects on the pharmokinetics of many drugs providesinformation for diagnosis and pharmacological treatment options for manydifferent diseases. There are currently over 1.8 million identifiedSNPse.

For the purpose of detection of SNPs, oligonucleotide primers may beemployed of length n designed to hybridise to a target DNA wherebyoccurrence of a particular nucleotide at position n+1 in the target, aposition of allelic variation, can be detected by providingcomplementary nucleotide for that position (either a dNTP or ddNTP)under conditions permitting extension of the primer by DNA polymerase.Such extension performed at or close to the surface of an ISFEToperating in accordance with the invention may be observed as a changein signal associated with proton release consequent upon nucleotideinsertion.

Where a SNP needs to be determined, e.g. the type of nucleotidesubstitution, then four separate reaction mixtures may be presented tothe sensing apparatus each containing template strands incorporating theSNP, but each providing a different possible nucleotide for insertion(A, T, C or G). Such SNP identification is illustrated by FIGS. 6 to 10.

FIGS. 6 and 8 illustrate determination of a homozygous SNP by singlenucleotide primer extension where all template strands derived from aregion of genomic DNA, e.g. by nucleic acid amplification, incorporatethe same SNP substitution. In this case, only one reaction mixture isobserved to cause pH drop associated with proton release detectable bythe ISFET. Four reaction mixtures were employed each containing adifferent dNTP (dTTP, dGTP, dATP or dCTP). Only the reaction mixtureproviding cytosine (C) produced a signal drop corresponding with singlenucleotide extension of primer indicating that the nucleotide at thepre-primed position on both DNA template strands is the nucleotidecomplementary to C, namely guanosine (G).

Individuals may possess a 50:50 mix of two bases at a particular DNAsite, i.e. be heterozygous at the position. FIGS. 7 and 9 illustratedetermination of an heterozygous SNP employing an ISFET in accordancewith the invention where two different bases are present at the relevantgenomic position and are identified by monitoring single nucleotideprimer extension on corresponding template strands in separate reactionmixtures each containing a different dNTP. Again four reaction mixtureswere employed each containing a different dNTP. Two reaction mixturesproduced a pH drop corresponding with single nucleotide extension byinsertion of different nucleotides to each primer. These were thereaction mixtures providing cytosine and guanosine indicating thepresence of their complementary counterparts at the pre-primed positionsof the DNA template strands. If the magnitude of the drops are compared,the two drops are roughly equal and the sum of the drops roughly equalto that seen with modelling of homozygous SNP detection.

Such monitoring of primer extension may similarly be employed toidentify a deletion allelic variant as illustrated by FIG. 10, or aninsertion allelic variant. With reference to the signal outputs of FIG.10 used to identify a deletion, again an oligonucleotide primer wasemployed of length n to target a defined position in target DNA at whichthe deletion occurs (position n+1 in the complementary target strandwith hybridised primer). Four reaction mixtures were provided containingprimer, DNA template incorporating the deletion, and one of each of thefour dNTPs. An ISFET output drop occurred in the chamber providingadenine (A) indicating the base thymine (T) to be present at the firstnucleotide position in the template after the 3′ end of the primer. Itwas possible to deduce that this accorded with a deletion by referenceto comparative sequence information.

Detection of allelic variants in amplified target regions of genomic DNAmay also be achieved by using an oligonucleotide probe specific for thevariant of interest such that it hybridises to the target DNA at thesite of the variant if present. Such allele specific hybridisation mayalso be identified by detecting single base extension of the probe byISFET monitoring of consequent proton release. Such allele specifichybridisation may be utilised in determining both point and moreextensive mutations, e.g. deletions of more than one base pair, fordisease diagnosis.

For ISFET monitoring of single base extension of a primer or variantspecific probe, e.g. for determination in amplified DNA of adisease-linked variant allele, the ISFET will be presented to thereaction mixture for DNA hybridisation and extension in a low volume,e.g. 50 μl or less, chamber or well, e.g. provided in apolydimethylsiloxane (PDMS) plate. Such apparatus is illustrated in FIG.5 and exemplification of use of such apparatus for single base extensionmonitoring is given in Example 1 below.

With reference to FIG. 5, the ISFET (16) and a silver/silver chloridereference electrode (17) are both provided in a low-reaction volumechamber (18) in a polydimethylsiloxane (PDMS) plate (19) with thereaction chamber having a covering (20) of PDMS in which is provided aninlet (21) for reagent addition. Above the covering is a seal (22)through which connections to instrumentation run protected by tubing(23) connecting with gate (G), source (S) and drain (D) terminals of theISFET. The output of the ISFET was measured using constant chargesource-follower instrumentation which monitored pH by keeping the ISFETgate voltage and drain current constant and recording changes ingate-source voltage, corresponding to the pH-dependent change in theISFET's flatband voltage. By placing the ISFET in a thermostatedwaterbath, single nucleotide extension of an oligonucleotide primer on aDNA target may be monitored at 37° C. or close to 37° C.

The low volume reaction chamber housing the ISFET may be a microfluidicchamber of a microfluidic device or cartridge. Incorporation of an ISFETinto a PDMS microfluidic chamber may be achieved by curing the PDMS withthe ISFET at 60° C. or less for no more than 4 hours. The desiredmicrofluidic chamber may then be created around the IFSET, e.g. bymanually removing PDMS in the region of the IFSET sensing region. TheISFET may be embedded in a horizontal plane at the bottom of a lowvolume reaction chamber, which is provided with microchannels for sampledelivery. Such a device is shown schematically in FIG. 11 with the ISFET(24) embedded in the base (25) of a low reaction volume chamber (26) ofless than one n1 (typical dimensions 100 μm×100 μm×10 μm). A number ofsuch ISFET-containing chambers may be provided in a single microfluidicchip. Means may be provided whereby target DNA of a single sample may bedelivered to more than one such chamber for simultaneous testing formore than one variation. The ISFET, housed in a microfluidic chamber,may be an integral part of a chip such as a silicon chip with resistiveon-chip heating elements and temperature sensors to control thetemperature for DNA hybridisation and extension. For monitoring ofsingle base primer/probe extension, the temperature of the reactionmixture will desirably be maintained constant at the optimal temperaturefor the DNA polymerase and thereby DNA extension.

Where the DNA sample is liable to contain both target DNA and unwantedbackground DNA, immobilisation of DNA probe or primer capable ofhybridising to the target is required to separate target DNA frombackground DNA. For this purpose, the DNA primer or probe for DNAextension monitoring may be immobilised on beads which are brought intothe vicinity of the ISFET sensing surface or linked to the ISFETdirectly or indirectly. In either case, immobilisation of the probe orprimer will be such as to enable the required separation step for targetDNA with washing to remove unwanted DNA. Ensuring close proximity of theDNA probe or primer to the ISFET sensing surface by such immobilisationalso has the benefit of increasing signal to noise ratio by localisingthe pH changes caused by the chain extension reaction close to thatsurface.

Thus, DNA extension monitoring in accordance with the invention maypreferably be of DNA primer or probe extension occurring on beads asillustrated by FIG. 12. The oligonucleotide primer or probe isimmobilized on the beads such that it will hybridise and thereby capturetarget DNA in the reaction chamber. If a dNTP complementary to thetarget template strand immediately after the 3′ end of the primer isprovided in the reaction mixture under DNA extension conditions, then achange in the ISFET output will be observed indicative of proton releaseas result of nucleotide addition to the primer. Use of beads may beadvantageously combined with use of an ISFET lying in a horizontal planeat the bottom of the reaction chamber as shown in FIG. 11 such that thebeads settle in the vicinity of the ISFET sensing surface. The beads maybe chosen such that gravitational settlement alone brings the beads intothe vicinity of the ISFET sensing surface. Alternatively,magnetic/metallic beads may be employed and magnetically drawn into thevicinity of the ISFET sensing surface. The beads may be sphericalparticles synthesised from any suitable material for the attachment ofDNA, e.g. silica, polystyrene, agarose or dextran. The size of the beadsmay be adjusted to assist gravitational settling. The beads can bewashed off the sensor surface using water or buffer solution. Linkage ofDNA primer or probe to the beads may be achieved using conventionalmethods, e.g. functionalisation of the bead surface. A spacer may beemployed. The coverage of the bead is controlled by adjusting the DNA tobead ratio. For example silica beads (e.g. about 200 nm diameter) may beemployed and DNA directly immobilized on the beads or immobilizedfollowing modification of the beads to provide a carboxylic functionalgroup. Plastic beads (e.g. plastic microbeads of about 1 μm) may forexample alternatively conveniently be employed.

As an alternative to the use of beads, as indicated above DNA primer orprobe for capture of target DNA may be linked directly or indirectly tothe ISFET whereby nucleotide extension is detected by the ISFET sensingsurface in the presence of target DNA. Provision of DNA primer or probeimmobilised on the ISFET may employ techniques well known for DNA probeimmobilisation on a solid surface, e.g. such techniques well known fromthe DNA microarray art. Thus, DNA probe or primer immobilisation on theISFET may be achieved by in situ-oligonucleotide synthesis (bylithography or other means).

The following references provide additional background informationrelevant to the invention:

Shakhov and Nyrén, ‘A Sensitive and Rapid Method for Determination ofPyophosphate Activity’, Acta Chem. Scand. B 36 (1982) 689-694;

R. Buck, ‘Electrochemistry of Ion-Selective Electrodes’, Sensors andActuators (1981) 1, 197-260;

Victorova et al, ‘New substrates of DNA polymerases’, FEBS Let. (1999)453, 6-10; and

Hanazato et al., ‘Integrated Multi-Biosensors Based on an Ion-sensitiveField-Effect Transistor Using Photolithographic Techniques’, IEEETransactions on Electron Devices (1989) 36, 1303-1310.

The following example provides fuller details of use of an ISFET sensingapparatus in accordance with the invention to monitor single base primerextension.

EXAMPLE 1

ISFET Monitoring of Single Base Primer Extension

The ISFET output from a reaction where there is an expected singlenucleotide base incorporated was monitored and compared to the ISFEToutput from a non-expected base incorporation signal. A single ISFETwith a silver/silver chloride reference electrode was used and the pHchange was measured in a very weakly buffered 50 μl reaction volume(FIG. 5). The output of the ISFET was measured using constant chargesource-follower instrumentation which monitored pH by keeping the ISFETgate voltage and drain current constant and recording changes ingate-source voltage, corresponding to the pH-dependent change in theISFET's flatband voltage.

Single-stranded oligonucleotides (5′-ACATCTGAGTCTGTAGTCTA-3′; SEQ IDNO:1) were purchased from MWG-Biotech. A 1 nmol/μl of oligonucleotidewas annealed with a slight excess of primer (5′-TAGACTAC-3′) and 5 μlwas added to a reaction mixture containing 1 mM NaCl and 2.5 mM MgCl2.

DNA polymerase (Klenow fragment, 3′ exonuclease deficient) was obtainedfrom Amersham Biosciences. 0.5 μl of this Klenow fragment was added tothe reaction mixture to produce a final enzyme concentration of 0.1units/μl. dNTP, which was either complementary (adenosine, FIG. 14) ornon-complementary (FIG. 15) for the position on the templateoligonucleotide immediately following the 3′ end of the hybridisedprimer, was mixed with the enzyme just prior to its addition and theenzyme/dNTP mixture added to the reaction chamber to trigger anextension reaction. The final dNTP concentration was 1 mM. The start pHof the DNA mixture was adjusted to 7.0 using small quantities of NaOHand HCl. All reactions were conducted at 37.1° C. by placing the ISFETin a thermostated waterbath and the ISFET output was recorded using acommercial 12-bit analogue to digital converter (PICO Technologies Ltd.)connected to a laptop computer.

The above procedure was repeated for all four nucleotides.

It was observed that after the addition of the complementary nucleotide(dATP), ISFET voltage output decreased significantly from baselinesteady-state ISFET output (FIG. 14) whereas for addition ofnon-complementary nucleotides (FIG. 15), the ISFET output signal diddeviate, but tended back towards the baseline signal. Therefore, thesignificantly lower ISFET signal endpoint for complementary nucleotideaddition, represented proton release through single nucleotide extensionof the hybridised primer on the template oligonucleotide.

Repetition of experiments for both complementary and non-complementarydNTPs showed that deviations from baseline ISFET signal output uponaddition of the enzyme/dNTP mixture can occur in either direction,possibly due to adsorption of DNA, agitation, adsorption of Klenowfragment, ISFET signal drift etc. Therefore when using this method todetermine which of the four nucleotides added has been involved in chainextension, a differential comparison of all four ISFET outputs isrequired to eliminate background effects such as these and determinewhich nucleotide addition has given a significant and steady deviationfrom baseline.

As a variation of the method above, it will be appreciated that adifferential arrangement may be employed with a platinum electrode andtwo ISFETs, one of which is insensitive to the reaction being monitored.Other possible variations will be immediately apparent to those skilledin the art.

The invention claimed is:
 1. A sensing apparatus comprising at least twoion sensitive field effect transistors (ISFET) and at least two sets ofreagents; a first of the transistors exposable to an unknown sample anda first of the sets of reagents, and separated from a second of thetransistors exposable to an unknown sample and a second of the sets ofreagents, different from the first set of reagents; and furthercomprising means for determining the difference between electricalsignals generated by the first and second transistors, to provide anoutput signals to identify the sample.
 2. The sensing apparatusaccording to claim 1, further comprising microfluidic chambers having avolume of less than 50 μL.
 3. The sensing apparatus according to claim2, wherein each ISFET is housed within the microfluidic chamber toreceive the unknown sample and one of the sets of reagents.
 4. Thesensing apparatus according to claim 1, further comprising microchannelsto deliver the unknown sample.
 5. The sensing apparatus according toclaim 4, further comprising a plate covering a silicon chip integratingthe ISFETs, the plate having portions removed to provide saidmicrofluidic chambers.
 6. The sensing apparatus according to claim 5,further comprising a heating element and a temperature sensor integratedinto the silicon chip.
 7. The sensing apparatus according to claim 2,further comprising means to deliver the sample to more than one of saidmicrofluidic chambers for simultaneous testing of more than onevariation of the sample.
 8. The sensing apparatus according to claim 2,further comprising a seal for the microfluidic chambers.
 9. The sensingapparatus according to claim 1, wherein the unknown sample comprises atarget nucleic acid.
 10. The sensing apparatus according to claim 1,wherein the sets of reagents comprise allele specific reagents.
 11. Thesensing apparatus according to claim 9, wherein the sets of reagentscomprise one or more types of nucleotide and an oligonucleotide servingas a primer or probe for the target nucleic acid.
 12. The sensingapparatus according to claim 9, wherein one of the sets of reagents iscomplementary and one of the sets of reagents is non-complementary withthe target nucleic acid.
 13. The sensing apparatus according to claim 1,wherein each set of reagents comprise magnesium.
 14. The sensingapparatus according to claim 1, further comprising beads having probesor primers immobilised on the surface.
 15. The sensing apparatusaccording to claim 1, wherein the apparatus further comprises monitoringmeans arranged to monitor the time at which fluctuations occur in theelectrical signals generated by the first and second transistors and themagnitude of said fluctuations.
 16. The sensing apparatus according toclaim 1, wherein the first and second transistors are connected to firstand second operational amplifiers respectively.
 17. The sensingapparatus according to claim 1, wherein the first and second transistorsare arranged to form part of first and second operational amplifiersrespectively.
 18. The sensing apparatus according to claim 1, wherein anelectrical signal is passed to an electrode to maintain voltages appliedto the first and second transistors at a constant level.